The focus of this proposal is to study the relationship between sphingolipid synthesis and sterol regulatory element binding-proteins (SREBP), key transcription factors of lipid metabolism. SREBP and sphingolipid related pathways contribute significantly to the morbidity related to cardiovascular and neurodegenerative diseases and are thus specifically relevant to the aging population. Cholesterol and sphingomyelin are independent risk factors for coronary artery disease. We recently showed that sphingolipid synthesis correlates with SRE-mediated gene transcription. Current data show that serine-palmitoyl transferase (SPT), the rate-limiting enzyme of sphingolipid de-novo synthesis is essential in the feedback regulation of SREBP. Together, these mechanisms are likely relevant in hereditary sensory neuropathy (HSN), caused by mutations in a subunit of SPT. Our data in HSN cells demonstrate increased SRE-mediated gene transcription, cholesterol synthesis and mass and indicate altered cellular cholesterol transport. We demonstrate that sphingolipid mediated regulation of SREBP-related pathways differs significantly in neuronal cells. We also demonstrate that diacylglycerol, a metabolite of sphingolipid synthesis, affects the cellular trafficking of SREBP. The central hypothesis is that sphingolipid synthesis is critical in the regulation of SREBP metabolism. The overall goal of the project is to investigate mechanism(s) how sphingolipid synthesis affects SREBP metabolism. We propose to develop fibroblasts and neuronal cell lines in which sphingolipid synthesis can be systematically modified using inducible siRNA and overexpression strategies of key enzymes. We will also assess the role of inhibitors and feedback regulators of sphingolipid synthesis on the regulation of SREBP-related lipid metabolism (aim 1). We will use fluorescent confocal microscopy to investigate the role sphingolipid synthesis and metabolites of sphingolipid synthesis on SREBP trafficking (aim 2). Results are expected to identify novel regulatory mechanisms that contribute to the pathogenesis of cardiovascular and neurodegenerative diseases and are amenable to therapeutic intervention. We identified a pathway that regulates important risk factors for cardiovascular and neurodegenerative diseases. We propose to further investigate this pathway to identify mechanisms that could be novel therapeutic targets - a desirable therapeutic goal in the interest of public health.